Cytochrome P450 proteins (CYP(s), or alternatively P450(s)) are a family of enzymes involved in the oxidative metabolism of both endogenous and exogenous compounds. P450 enzymes are widely distributed in the liver, intestines and other tissues (Krishna et al., Clinical Pharmacokinetics. 26:144-160, 1994). P450 enzymes catalyze the phase I reaction of drug metabolism, to generate metabolites for excretion. The classification of P450s is based on homology of the amino acid sequence (Slaughter et al The Annals of Pharmacotherapy 29:619-624, 1995). In mammals, there is over 55% homology of the amino acid sequence of CYP450 subfamilies. The differences in amino acid sequence constitute the basis for a classification of the superfamily of cytochrome P450 enzymes into families, subfamilies and isozymes.
When bound to carbon monoxide (CO), the CYP proteins display a maximum absorbance (peak) at 450 nm in the visible spectra, from which its name, cytochrome P450 is derived (Omura et al., J. Biol. Chem. 239:2370, 1964). The proteins contain an iron cation and are membrane bound enzymes that can carry out electron transfer and energy transfer. Over 200 genes encoding cytochrome P450 proteins have been identified. Those genes have been divided among more than 30 gene families, which are organized into subfamilies that vary in regulation of gene expression and in amino acid sequence homology, substrate specificity, catalytic activity, and physiological role of the encoded enzymes.
Representative cytochrome P450 (CYP) genes and examples of the known substrates of CYP proteins encoded by those genes are discussed below. See also the discussion in Klassen, ed., Casarett and Doull's Toxicology: The Basic Science of Poisons, McGraw-Hill, 1996, pp. 150 ff. Further information about cytochrome P450 substrates, can be found in Gonzales and other review articles cited above. Current information sources available via the Internet include the “Cytochrome P450 Homepage”, maintained by David Nelson, the “Cytochrome P450 Database”, provided by the Institute of Biomedical Chemistry & Center for Molecular Design, and the “Directory of P450-containing Systems”, provided by Kirill N. Degtyarenko and Peter Fabian.
CYP1A1: diethylstilbestrol, 2- and 4-hydroxyestradiol
CYP1A2: acetaminophen, phenacetin, acetanilide (analgesics), caffeine, clozapine (sedative), cyclobenzaprine (muscle relaxant), estradiol, imipramine (antidepressant), mexillitene (antiarrhythmic), naproxen (analgesic), riluzole, tacrine, theophylline (cardiac stimulant, bronchodilator, smooth muscle relaxant), warfarin.
CYP2A6: coumarin, butadiene, nicotine
CYP2A13: nicotine
CYP2B1: phenobarbital, hexobarbital
CYP2C9: NSAIDs such as diclofenac, ibuprofen, and piroxicam; oral hypoglycemic agents such as tolbutamide and glipizide; angiotensin-2 blockers such as irbesartan, losartan, and valsartan; naproxen (analgesic); phenytoin (anticonvulsant, antiepileptic); sulfamethoxazole, tamoxifen (antineoplastic); torsemide; warfarin, flurbiprofen
CYP2C19: hexobarbital, mephobarbital, imipramine, clomipramine, citalopram, cycloguanil, the anti-epileptics phenytoin and diazepam, S-mephenytoin, diphenylhydantoin, lansoprazole, pantoprazole, omeprazole, pentamidine, propranolol, cyclophosphamide, progesterone
CYP2D6: antidepressants (imipramine, clomipramine, desimpramine), antipsychotics (haloperidol, perphenazine, risperidone, thioridazine), beta blockers (carvedilol, S-metoprolol, propafenone, timolol), amphetamine, codeine, dextromethorphan, fluoxetine, S-mexiletine, phenacetin, propranolol
CYP2E1: acetaminophen; chlorzoxazone (muscle relaxant), ethanol; caffeine, theophylline; dapsone, general anesthetics such as enflurane, halothane, and methoxyflurane; nitrosamines
CYP3A4: HIV Protease Inhibitors such as indinavir, ritonavir, lopinavir, amprenavir, tipranavir, darunavir, and saquinavir; HIV integrase inhibitors such as raltegravir, Hepatitis C virus (HCV) protease inhibitors, benzodiazepines such as alprazolam, diazepam, midazolam, and triazolam; immune modulators such as cyclosporine; antihistamines such as astemizole and chlorpheniramine; HMG CoA Reductase inhibitors such as atorvastatin, cerivastatin, lovastatin, and simvastatin; channel blockers such as diltiazem, felodipine, nifedipine, nisoldipine, nitrendipine, and verapamil; antibiotics such as clarithromycin, erythromycin, and rapamycin; various steroids including cortisol, testosterone, progesterone, estradiol, ethinylestradiol, hydrocortisone, prednisone, and prednisolone; acetaminophen, aldrin, alfentanil, amiodarone, astemizole, benzphetamine, budesonide, carbamazepine, cyclophosphamide, ifosfamide, dapsone, digitoxin, quinidine (anti-arrhythmic), etoposide, flutamide, imipramine, lansoprazole, lidocaine, losartan, omeprazole, retinoic acid, FK506 (tacrolimus), tamoxifen, taxol and taxol analogs such as taxotere, teniposide, terfenadine, buspirone, haloperidol (antipsychotic), methadone, sildenafil, trazodone, theophylline, toremifene, troleandomycin, warfarin, zatosetron, zonisamide.
CYP6A1: fatty acids.
The efficacy of a drug can be dramatically affected by its metabolism in the body. In addition, the failure to maintain therapeutically effective amounts of a drug may also impact its long term efficacy. This situation may arise particularly in treatment of infectious diseases, such as viral or bacterial infections, where the inability to maintain an effective therapeutic dose can lead to the infectious agent(s) becoming drug resistant. To avoid the consequences of metabolism and sustain a therapeutically effective amount of drugs that are rapidly metabolized in a subject, or a specific tissue of a subject, the drugs often must be administered in a sustained release formulation, given more frequently and/or administered in higher dose than more slowly metabolized drugs administered by the same routes.
A common pathway of metabolism for drugs containing lipophilic moieties is via oxidation by one or more CYP enzymes. The CYP enzyme pathway metabolizes many lipophilic drugs to more polar derivatives that are more readily excreted through the kidney or liver (renal or biliary routes). That pathway renders many compounds having strong biological efficacy that would otherwise be potentially powerful therapeutics essentially useless by virtue of their rapid metabolism, which results in short half-lives in vivo, particularly where drugs are administered by the oral route.
Poor bioavailability, particularly oral bioavailability, due to first pass CYP metabolism, which leads to elimination of drugs via the liver and/or intestinal routes, is a major reason for the failure of many drug candidates in clinical trials. Where extensive metabolism by intestinal CYP occurs, first pass metabolism can lead to poor drug absorption from the GI tract. Similarly, extensive hepatic CYP metabolism can result in low circulating (plasma or blood) levels of a drug.
Alteration in drug metabolism by CYP proteins may have undesired or unexpected consequences. In some instances, metabolic by-products of CYP enzymes are highly toxic and can result in severe side effects, cancer, and even death. In other instances, alterations in CYP metabolism due to the interaction of agents may produce undesirable results.
Some examples of drug metabolism by CYP proteins and the effects of other agents on the metabolites produced by CYP proteins include:
Acetaminophen: Ethanol up-regulates CYP2E1, which metabolizes acetaminophen to a reactive quinone. This reactive quinone intermediate, when produced in sufficient amounts, causes liver damage and necrosis.
Sedatives: The sedative phenobarbital (PB) up-regulates several P450 genes, including those of the CYP2B and CYP3A subfamilies. Up regulation of these enzymes increases the metabolism and reduces the sedative effects of PB and the related sedative hexobarbital.
Antibiotics: The antibiotics rifampicin, rifampin, rifabutin, erythromycin, and related compounds are inducers of the CYP3A4 gene and are substrates of the enzyme product.
Anti-cancer agents: Taxol and taxotere are potent anti-cancer agents. Both drugs are extensively metabolized by CYP3A4 and have poor oral bioavailability. These drugs are only efficacious in parenteral formulations which, due to their poor solubility properties, are highly noxious to patients.
Nicotine: CYP2A6 and 2A13 convert nicotine, a non-toxic component of cigarette smoke, into NNK, a highly potent carcinogen that contributes to lung cancer from smoking.
Oral contraceptive/estrogen replacement therapy: Estrogens and estradiols are the active ingredients in oral contraceptives and in hormonal replacement therapies for post-menopausal women. Women who are also taking antibiotics such as rifampicin or erythromycin, or glucocorticoids such as dexamethasone, or who smoke, risk decreased efficacy of the estrogen/estradiol treatments due to increased metabolism of these compounds by up-regulated CYP3A4 and/or CYP1A2 enzymes.
Dextromethorphan: CYP2D6 metabolizes dextromethorphan to dextrorphan. Individuals who express high levels of CYP2D6 (so-called rapid metabolizers) do not receive therapeutic benefits from dextromethorphan due to extensive first-pass metabolism and rapid systemic clearance.
Protease Inhibitors: Protease inhibitors and non-nucleoside reverse transcriptase inhibitors currently indicated for use in treatment of HIV or HCV are typically good substrates of cytochrome P450 enzymes; in particular, they are metabolized by CYP3A4 enzymes (see e.g., Sahai, AIDS 10 Suppl 1:S21-5, 1996) with possible participation by CYP2D6 enzymes (Kumar et al., J. Pharmacol. Exp. Ther. 277(1):423-31, 1996). Although protease inhibitors are reported to be inhibitors of CYP3A4, some non-nucleoside reverse transcriptase inhibitors, such as nevirapine and efavirenz, are inducers of CYP3A4 (see e.g. Murphy et al., Expert Opin. Invest. Drugs 5/9: 1183-99, 1996).
Human CYP isozymes are widely distributed among tissues and organs (Zhang et al., Drug Metabolism and Disposition. 27:804-809, 1999). With the exception of CYP1A1 and CYP2A13, most human CYP isozymes are located in the liver, but are expressed at different levels (Waziers J. Pharmacol. Exp. Ther. 253: 387, 1990). A solution to the problem of drug degradation and first-pass metabolism is to control the rate of drug metabolism. When the rates of drug absorption and metabolism reach a steady state, a maintenance dose can be delivered to achieve a desired drug concentration that is required for drug efficacy. Certain natural products have been shown to increase bioavailability of a drug. For example, the effect of grapefruit juice on drug pharmacokinetics is well known. See Edgar et al., Eur. J. Clin. Pharmacol. 42:313, (1992); Lee et al., Clin. Pharmacol. Ther. 59:62, (1996); Kane et al., Mayo Clinic Proc. 75:933, (2000). This effect of grapefruit juice is due to the presence of natural P450-inhibiting components. Other compounds also have been used for inhibition of P450. For example, the HIV-1 protease inhibitor Ritonavir® is now more commonly prescribed for use in combination with other, more effective HIV protease inhibitors because of its ability to “boost” those other compounds by inhibiting P450-mediated degradation.
Present methods of inhibiting cytochrome P450 enzymes are not wholly satisfactory because of toxicity issues, high cost, and other factors. For example, using ritonavir to inhibit cytochrome P450 is not desirable in disorders other than HIV infection. It is apparent, therefore, that new and improved methods of inhibiting cytochrome P450 enzymes are greatly to be desired. In particular, methods where an inhibitor can be co-administered with another biologically active compound (e.g., a drug) that is metabolized by cytochrome P450 enzymes are highly desirable.